Spermatogenesis and the regulation of Ca(2+)-calmodulin-dependent protein kinase IV localization are not dependent on calspermin

Abstract

Calspermin and Ca(2+)-calmodulin-dependent protein kinase IV (CaMKIV) are two proteins encoded by the Camk4 gene. CaMKIV is found in multiple tissues, including brain, thymus, and testis, while calspermin is restricted to the testis. In the mouse testis, both proteins are expressed within elongating spermatids. We have recently shown that deletion of CaMKIV has no effect on calspermin expression but does impair spermiogenesis by disrupting the exchange of sperm basic nuclear proteins. The function of calspermin within the testis is unclear, although it has been speculated to play a role in binding and sequestering calmodulin during the development of the germ cell. To investigate the contribution of calspermin to spermatogenesis, we have used Cre/lox technology to specifically delete calspermin, while leaving kinase expression intact. We unexpectedly found that calspermin is not required for male fertility. We further demonstrate that CaMKIV expression and localization are unaffected by the absence of calspermin and that calspermin does not colocalize to the nuclear matrix with CaMKIV.

FIG. 1

Targeted deletion of calspermin. (A) Map of the Camk4 129/Sv genomic clone and construction of the targeting construct. The minimal calspermin promoter, which contains two CRE motifs, and the testis-specific exon (Ts) were replaced by neomycin (Neo) and thymidine kinase (TK) selectable markers flanked by loxP sites. Correctly targeted ES cells were transfected with Cre recombinase to delete the selectable markers, leaving a single loxP site. The probe used for Southern blot analysis is indicated by a black bar. (B) Southern blot analysis of KpnI(K)-digested genomic DNA from targeted and wild-type ES cells hybridized with the 5′ probe. The wild-type allele is 7 kb, while the mutant allele is 13 kb. (C) Northern blot analysis of total RNA probed with a portion of the calspermin cDNA, which hybridizes to both the 2.1-kb Camk4 mRNA and the 1.1-kb calspermin mRNA. Equal amounts (10 μg) of RNA were loaded per lane. (D) Western blot analysis of thymus extracts from wild-type (+/+), heterozygous (+/−), and homozygous null (−/−) animals detected with anti-CaMKIV antibody.

FIG. 2

CaSKO mice are fertile. (A) Testis weights of wild-type (+/+) and CaSKO (−/−) mice. (B) Sperm counts from wild-type (+/+) and CaSKO (−/−) mice. n = 6 for each genotype. Values are ± the standard deviation.

FIG. 3

Histology of CaSKO seminiferous tubules. (A and B) Histological analysis of wild-type (A) and CaSKO (B) testes. Magnification, ×170. (C and D) CaMKIV localization is not dependent on calspermin. Testis sections from wild-type (C) and CaSKO (D) mice were analyzed by immunohistochemistry as described in Materials and Methods. CaMKIV expression patterns are unchanged in CaSKO testes Magnification, ×85.

FIG. 4

CaMKIV expression and targeting to the nuclear matrix are unaltered in CaSKO mice. (A) Western blot analysis of testes extracts blotted with anticalspermin, which detects both CaMKIV and calspermin. (B) Soluble and nuclear matrix preparations from wild-type (+/+), heterozygous (+/−), and CaSKO (−/−) testes were immunoblotted for CaMKIV. (C) Testes extracts from these mice were also immunoblotted with an antibody against calmodulin.